JPH0843389A - Immunological measuring method for serum amyloid-a - Google Patents

Abstract

PURPOSE:To suppress variation of the measurement from reagent lot to lot by improving the linearity in the dilution in an immunological measuring of a serum amyloid-A (SAA) which serves as a marker for inflammation etc. CONSTITUTION:A high density lipoprotein fraction (HDL) is added to the field of immune reaction of an anti-SAA antibody wiith SAA, and thereby an immunological measuring of the SAA is made. This can improve the diluting linearity economically by a simple method and also suppress variation of the measurement between reagent lots.

Description

Detailed Description of the Invention

[0001]

The present invention relates to amyloid A in serum.
The present invention relates to an immunological assay method (hereinafter, abbreviated as SAA). SAA is a precursor protein of amyloid protein A (hereinafter abbreviated as AA protein) that deposits in tissues in certain types of amyloidosis, and has a molecular weight of about 12,000.
Is a serum protein of. (J.Clin.Invest.53: 1054-1061,197
4) In recent years, it has been revealed that the serum level of this SAA is elevated in inflammatory diseases other than amyloidosis, and it has been evaluated as a sensitive inflammatory marker. (Clinical examination 32:
2, P168,1988)

[0002]

2. Description of the Related Art In the field of clinical examination and the like, immunological measurement is often used in order to easily measure a substance. SAA is no exception, and radioimmunoassay (hereinafter abbreviated as RIA) and enzyme immunoassay (Scand.J.Im
munol.18: P329,1983, hereinafter abbreviated as EIA), immunoprecipitation assay (Marker Proteins in Inflammation, 3: P157,1)
986, hereinafter abbreviated as TIA), etc., and many reports on immunological assay methods. With these assays, purified SA
The measurement is carried out by reacting an antibody obtained with A as an immunogen with SAA. In other words, general immunological measurement techniques are simply applied to SAA. These conventional techniques have the following problems.

1. Poor dilution linearity When immunologically measuring SAA, good dilution linearity can be obtained when the concentration is relatively low. But for example 3
With a high-value sample exceeding 00 μg / ml, the dilution linearity varies. In other words, there are cases where the required dilution linearity cannot be obtained depending on the sample or the reagent lot. Dilution linearity means that the sample is 3/5
It means that the measured value becomes 3/5 when diluted to 1, and the measured value becomes 1/5 when diluted to 1/5. Therefore, if you draw a graph with a dilution ratio on the horizontal axis and a measurement value on the vertical axis for a measurement system with good dilution linearity,
The graph becomes a straight line passing through the origin 0. On the contrary, when the dilution linearity is insufficient, there are cases in which this graph does not become a straight line but becomes an upward warped curve, for example.
The reactivity of such a specimen with the sample is different from that of the standard substance, and therefore the reliability of the measured value obtained from the calibration curve using the same standard is lost. In other words, it is necessary to select a standard that has the same reactivity (specificity) as all samples and shows good dilution linearity. However, it is actually impossible to predict in advance what kind of reactivity all samples will show with the standard and guarantee the quality. A sample containing a high concentration of SAA cannot be accurately measured unless diluted (antigen excess region). However, when measuring diluted samples with conventional techniques,
Poor dilution linearity causes variations in measured values. Further, there is a problem in obtaining a calibration curve using a standard sample. Since the linearity of dilution is poor, the calibration curve may not be linear and the measurement range may be limited. If a multi-concentration standard sample is prepared in advance, the problem of dilution linearity can be avoided at least in the preparation of the calibration curve. However, preparing a large number of standards may cause an increase in reagent cost, and it cannot be overemphasized that the reliability of the calibration curve obtained under conditions different from the actual analytical sample is low.

[0004] 2. Lot difference easily occurs In the conventional measurement method, when the measured values of reagents of different lots of the same sample are compared, the values tend to fluctuate. In a small-scale use in a research facility, the once-prepared reagent can be used for a considerable period of time, so the problem of lot difference is hard to be exposed. However, when the immunological measurement reagents are supplied on a commercial basis, a large lot difference is a fatal problem. The present inventors have confirmed that even when a calibration curve is prepared for each lot, for example, when the measurement value of the lot A is 100, it is about 80 for the lot B and about 120 for the lot C. , The measured values of multiple samples may be biased to the same tendency. Such a phenomenon is particularly remarkable when the sample is diluted, and thus becomes a serious problem in the measurement of a high-value sample that exceeds the measurement range of the reagent.

[0005]

The object of the present invention is to solve the problem of SA
It is to improve the dilution linearity in the immunoassay of A and to reduce the reagent lot difference.

[0006]

The present invention is firstly directed to "SA".
SAA characterized by adding a high-density lipoprotein fraction to the reaction system when reacting A with an anti-SAA antibody
Immunological assay method of ".

The high-density lipoprotein fraction of the present invention (hereinafter referred to as H
(Abbreviated as DL) may be derived from any animal even when measuring human SAA. Specifically, almost the same effect can be obtained with HDL of humans, horses, cows, goats, pigs and the like. As the HDL in the present invention, those purified from various animal sera may be used, or animal serum containing HDL may be added as it is. As HDL, commercially available HDL purified from human or animal blood may be used. Alternatively, since a method for separating and purifying HDL has already been established, it is possible to purify and use a necessary amount. What is HDL?
Specific gravity separated from serum by ultracentrifugation 1.063-
It is a generic term for lipoproteins having 1.210 g / ml. After that, lipoproteins composed of α-lipoprotein fractions electrophoretically electrophoresed in the α1 globulin region as well as ultracentrifugation, and fractions of lipoproteins that do not precipitate with heparin-Mn 2+ are also called HDL. Has become. According to the findings obtained so far, about 50 HDL of healthy people
% Consists of lipids and the remaining 50% consists of a protein called HDL apoprotein. Cholesterol (20%), phospholipids (23%), triglyceride (5%), etc. make up lipids, while apoproteins account for 90% of ApoA-I and A-II.

When HDL is not a pure product but in a crudely purified state or when animal serum is added as it is, it is preferable to confirm the HDL value in advance so that the addition amount can be controlled. Since animal serum contains SAA, it must be removed in advance. SAA is
It can be easily removed by immunological adsorption with the same antibody used for the measurement. However, some animal species do not immunologically intersect with the SAA to be measured. In such a case, it is not necessary to remove it because it is not affected by the coexisting SAA. For example, human SA
The antibody obtained using A as the immunogen hardly reacts with horse or bovine SAA. The amount of HDL used in the present invention is 1 ml of the reaction solution when the cholesterol of the added HDL is measured if a general serum sample is analyzed.
It is preferable to add at least about 0.3 μg or more. Of course, this concentration is a value when HDL separated from normal human serum or animal serum by ultracentrifugation is added. Since the average composition ratio of cholesterol in HDL is about 20%, the total amount of HDL can be about 1.5 μg / ml or more. For example, commercially available human purified HDL (protein 1 mg
(Including HDL-C 25 μg or more, made by SIGMA)
If HDL is used as HDL, the amount of HDL-C described above is about 10 times as much, that is, 3 μg /
More than ml is good. If the amount added is less than this concentration, sufficient effects may not be obtained. On the other hand, even if used in an unnecessarily large amount, there is no difference in the expected effect,
Also, when HDL is added as serum, HD contained in serum
Since there is a limit to the amount of L, there will inevitably be an upper limit.

For these reasons, a more practical amount of HDL used is about 1.5 to 2000 μg per ml of the reaction solution.
Can be shown. This value is the value of protein in HDL. 0.3 to 300 μg per 1 ml of reaction solution when cholesterol concentration is used as an index of HDL, 0.4 to 500 μg when phospholipid is used as an index, and ApoA-I is used as an index. The value is 0.5 to 600 μg, and the value is 0.2 to 200 μg when ApoA-II is used as an index. When cholesterol concentration is used as an index, the preferred concentration is 5 μg
As described above, if it is possible to obtain a reliable effect for a wider range of samples, it is preferable to set it to 10 μg or more. These values are calculated based on the average component ratio in human serum, and when obtaining materials for other animal species as HDL, set appropriate values based on the composition of HDL in each animal species. can do. Of course, when analyzing a sample containing almost no HDL, such as spinal fluid, it is preferable to set the amount of HDL added to a high level. Although HDL is a protein that is also present in normal serum, HDL derived from a sample has almost no effect in the SAA assay method of the present invention.

The immunological assay method of the present invention is RI
A, EIA, particle agglutination immunoassay, TIA and the like can be exemplified. Among these measurement methods, the particle agglutination immunoassay method and TIA are preferable measurement methods because they can be measured by a simple operation without using a special device and can be expected to have sufficient sensitivity and reproducibility. . The particle agglutination immunoassay measures the SAA concentration by optically following the SAA agglutination of inactive particles to which the anti-SAA antibody has been immobilized, and the TIA optically tracking the precipitate produced by the reaction between the anti-SAA antibody and SAA. . In RIA and EIA, S
A sandwich method in which AA is sandwiched with a plurality of antibodies and a competition method in which labeled SAA competes with the SAA to be measured for the antibody are possible. In any method, the effect of the present invention can be obtained by adding HDL to the place of immune reaction. The anti-SAA used in these measurement methods
The antibody may be a polyclonal antibody or may be a monoclonal antibody. Also, both may be used in combination. In particular, in RIA and EIA using a solid phase antibody, by combining a monoclonal antibody with a polyclonal antibody, it becomes possible to construct an excellent measurement system that simultaneously satisfies sensitivity and specificity. Although there have been some reports on SAA antibodies, known antibodies can be used in the immunological assay of the present invention. For example, there is a report by TLMc Donald et al. As a SAA monoclonal antibody (JI
mmunol.Methods, 144, 149-155; 1991). As is also reported in this report, in order to perform the sandwich method with a monoclonal antibody, it is necessary to prepare an antibody that recognizes a plurality of epitopes at physically separated positions on SAA.
The same applies to the particle agglutination reaction method and TIA, and when a monoclonal antibody is used, it must be allowed to react at a plurality of sites.

Secondly, the present invention is characterized in that "HDL is added to the reaction system in an immunological assay method in which SAA is reacted with an anti-SAA antibody and SAA is measured based on the degree of binding between the two. Method for suppressing fluctuations in the measured value. The present invention provides a method for suppressing fluctuations in measured values between reagent lots by adding HDL to the immunoreaction field based on the conditions as described above. The variation of the measured value between the reagent lots means that the same sample shows different values among the reagent lots when measured under the same conditions. According to a general reagent quality control method, it is determined that the required quality is satisfied if the value obtained by measuring the standard product or the pooled sample falls within a certain range. However, in the immunological measurement of SAA, there is a special tendency that a large lot difference occurs when other serum is used as a sample, even if there is no variation in the measured value between lots in a certain pool serum. It was confirmed that The present invention is such SAA
The present invention provides a method for effectively suppressing the variation in the measured value between the reagent lots, which is unique to the.

Thirdly, the present invention provides "a reagent for immunological measurement of SAA composed of an anti-SAA antibody and HDL". The immunological measurement reagent of the present invention can be applied to reagents for a wide range of measurement techniques as described in the measurement method of the present invention. Specifically, RI
It can be a reagent for A, EIA, particle agglutination immunoassay, TIA and the like. In RIA and EIA, a reagent is composed of components such as an anti-SAA antibody (or a second antibody) immobilized on a solid phase, a labeled anti-SAA antibody (or a labeled SAA), and a diluent for diluting a sample to an appropriate concentration. To be done. On the other hand, particle agglutination immunoassay or TIA
The reagent for is composed of a component in which an anti-SAA antibody is immobilized on insoluble particles (the free antibody remains as it is in TIA), an appropriate diluent and the like. The HDL of the present invention may be added to any of these reagent components. H for components containing antibodies
If DL is added, reagents composed of proteins can be grouped together, which is advantageous in adding preservatives and the like.
On the other hand, if a large amount of HDL is allowed to coexist with the antibody solution or the like, it may be added to the diluent if it may cause problems such as precipitation. In any case, even if HDL is not used, adding it to an essential component does not cause operational problems.

Anti-SA of immunoassay reagent according to the present invention
As the A antibody, any antibody can be used as described above. That is, it may be a polyclonal antibody or a monoclonal antibody. The antibody can also be used as a fragment digested with an appropriate enzyme for the purpose of suppressing nonspecific effects of rheumatoid factors and complement. Known antibody fragments include F (ab ′) 2 fragments derived from pepsin. The anti-SAA antibody can be formed into various forms depending on the intended measurement technique. That is, if it is RIA, it is RI, and also EI
In A, it is labeled with an enzyme. Alternatively, when it is used as a solid phase antibody, it is immobilized on the container wall, bead carrier or the like. In the case of particle agglutination immunoassay, it is immobilized on inert particles such as polystyrene latex, liposome, and gold colloid. Immobilization of the antibody on the solid phase or particles can be achieved by chemical bonding or physical adsorption. The SAA immunological assay reagent of the present invention may be combined with other known components. That is, a buffering agent that gives a pH necessary for an immune reaction, a reaction enhancer that promotes an immune reaction, a reaction stabilizer or a blocker that suppresses a non-specific reaction, and a preservative such as sodium azide that enhances the preservation of reagents are used. You may combine. The following may be used as the buffer. GOOD buffer 2-morpholinoethanesulfonic acid (2- (N-Morpholino) et
hanesulfonic acid, abbreviated as MES) Piperazine-bis (2-ethanesulfonic acid) (Piperazi
ne-N, N'-bis (2-ethanesulfonic acid), abbreviated as PIPES) (2-acetamido) -2-aminoethanesulfonic acid (N- (2-Acetamido) -2-aminoethanesulfonic acid, AC
Abbreviated as ES) Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (N, N-Bis (2-hydroxyethyl) -2-aminoehtanesulfo
nic acid, abbreviated as BES) Bis (2-hydroxyethyl) iminotris (hyd
roxymethyl) methane, abbreviated as Bis-Tris) 3- [bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (3- [N, N-Bis (2-hydroxyet
hyl) amino] -2-hydroxypropanesulfonic acid, DIPS
Abbreviated as O) 2-Hydroxyethylpiperazine-3-propanesulfonic acid (N-2-Hydroxyethylpiperazine-N'-3-propanesulfon)
nic acid, abbreviated as EPPS.) N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic a
abbreviated as CID and HEPES) 2-Hydroxyethylpiperazine-2-hydroxypropane-3-sulfonic acid (N-2-Hydroxyethylpiperazine-
N'-2-hydroxypropane-3-sulfonic acid, HEPPSO
3- (morpholino) propanesulfonic acid (3- (N-Morphol
ino) propanesulfonic acid, abbreviated as MOPS) 3- (N-Morpholino) -2-hydroxypropanesulfonic acid
, MOPSO) Piperazine-bis (2-hydroxypropanesulfonic acid) (Pioerazine-N, N'-bis (2-hydroxypropanesulfonic acid)
acid), abbreviated as POPSO) N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid
abbreviated as acid and TAPS) Tris (hydroxymethyl) methyl-2-hydroxy-
3-Aminopropanesulfonic acid (N-Tris (hydroxymethy
l) methyl-2-hydroxy-3-aminopropanesulfonic acid, T
Abbreviated as APSO) Tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid (N-Tris (hydroxymethyl) methyl-2-aminoeth
Anesulfonic acid, abbreviated as TES) Other buffering agents 2-Amino-2-hydroxymethyl-1,3-propanedio
l) Tris (hydroxymethyl) aminomethane (Tris (hydro
xymethyl) aminomethane) Phosphate buffer ammonium buffer Also among these buffers, GOOD buffers such as HEPES and PIPES not only give a pH favorable for immune reactions, but also have a small effect on proteins, As a preferable buffering agent. Polyethylene glycol and dextran sulfate are known as reaction enhancers. Further, the following are known as reaction stabilizers and blockers. That is, BSA, animal serum, Ig
G, IgG fragments (Fab and Fc), albumin, milk protein, amino acids, polyamino acids, polysaccharides such as choline and sucrose, gelatin, gelatin degradation products, polyhydric alcohols such as casein and glycerin react in immune reactions. It is known to be effective in stabilizing and suppressing non-specific reactions. The SAA immunological assay reagent of the present invention containing these various components can be supplied in a solution state or in a dry state. In order to distribute in solution, various surfactants, sugars, and
An inactive protein or the like may be added. These stabilizers are effective as stabilizers or excipients when the reagent is dried.

In the present invention, SAA contained in blood, cerebrospinal fluid, cord blood, etc. can be measured. As described above, SAA in blood serves as an index of inflammation. Although SAA is a substance that can be detected in mammals other than humans, the assay method of the present invention can also be applied to the SAA measurement of mammals other than humans.

[0015]

The HDL of the present invention has the effect of improving the dilution linearity and suppressing the fluctuation of the measured value between reagent lots by coexisting with the anti-SAA antibody and the SAA immunoreaction field. It is speculative about the mechanism by which HDL exerts such an action. It is already known that SAA exists in the blood in a state of being complexed with HDL, but there is no report on how it affects the immunological reaction. Although the mechanism of action of HDL in the present invention is unknown, the present inventors presume as follows. Since SAA exists in the living body in a state of being complexed with HDL, it is expected that the decrease in HDL concentration due to the dilution of the sample will lead to the desorption of SAA from HDL. Standard product is SA combined with HDL
Since it is in state A, the reaction specificity may differ from that of desorbed SAA. When HDL is added here, the HDL concentration does not decrease even when the sample is highly diluted, or the desorbed SAA associates with HDL again, and therefore the reaction specificity common to that of the standard product is obtained. It is possible to explain that the sex is maintained. On the contrary, when HDL is excessively present, the original SAA-H
Since it may affect the balance between DLs, HDL
It cannot be said that the replenishment of the above gives the effect of the present invention immediately. The effect of the present invention due to the addition of HDL cannot be easily obtained from the conventional knowledge.

The major feature of the present invention is that H
To add DL. HDL is a protein involved in the transport of lipids in blood, and is called a high specific gravity because it has a higher specific gravity than other lipid-related proteins.
As is clear from this feature, for example, when blood or serum is used as the measurement target, HDL derived from the sample was present in the immunoreaction field even in the conventional immunological measurement method. For example, in the serum of healthy people, 180
It is said that ~ 220 mg / dl of HDL is present (modern medicine, 12,523-529; 1980), and when this serum is analyzed under the conditions of the examples described later, about 10-1 is present in the reaction solution.
About 4 μg / ml of HDL (about 20% of this in HDL-C) is present. In the present invention. In addition to HDL derived from such a sample, artificial addition of HDL has realized improvement of dilution linearity and suppression of variation in measured values between reagent lots, which cannot be obtained by conventional methods. In addition, in an immunological reaction-based assay method, it is known that non-specific reactions can be effectively suppressed by adding normal animal serum to the reaction site. However, the animal serum in this case mainly functions by blocking non-specific adsorption, and thus the HD of the present invention is used.
It does not suggest the action of L. Further, the applicant is
A patent application has been filed for a technique that uses SAA combined with DL as a standard (Japanese Patent Laid-Open No. 4-254769). This patent is intended to solve the problems that occur when using purified SAA as a standard, and again does not suggest the action of HDL of the present invention.

[0017]

INDUSTRIAL APPLICABILITY The immunological assay method for SAA according to the present invention,
Alternatively, with the immunoassay reagent, it is possible to always perform measurement with good dilution linearity. The action of HDL of the present invention makes it difficult for the reactivity between SAA and the antibody to change. As a result, a constant dilution linearity can always be obtained without being affected by differences in the type of sample, the degree of dilution, the reagent lot, etc., and highly reliable measurement is possible. By improving the dilution linearity, a highly reliable value can be obtained even when a sample containing a high concentration of SAA is diluted and measured.

The immunological assay method for SAA of the present invention comprises:
It is possible to suppress the variation in the measured value between the reagent lots, which cannot be avoided by the conventional technique. The fluctuation of the measured value between the reagent lots is a fatal obstacle particularly when the reagent is supplied on a commercial basis, but the present invention solves this problem by an extremely simple method. In the present invention, since it is only necessary to add HDL, which is easily procured, as a reagent component, there is almost no effect on cost, and on the other hand, no special operation or condition is required in terms of operation. As described above, the present invention solves, by a simple method, a major problem that hinders the commercial immunoassay of SAA. Hereinafter, the present invention will be described in detail based on examples.

[0019]

【Example】

1. Reagent for immunological measurement of SAA 1-1. Purified SAA SAA-rich patient ascites pool (118 μg / ml) 1 L
First, the specific gravity is 1.23 g / l by ultracentrifugation.
The upper layer was sampled, then the lower layer having a specific gravity of 1.063 g / l was sampled, degreased with methanol / ether (1: 2) under cooling, and then Sephadex G-200 column (6M urea, 0.
0.01M Tris-HCl buffer containing 5% Tween 20
(Equilibration with H8.6), and then, by using a conventional method, sepharose 4B (Pharmacia) activated with bromocyan was passed through a column to which anti-ApoA-I, ApoCIII, and human serum albumin antibodies were bound to remove contaminant proteins. Then, 31 mg of purified SAA was obtained from 1 L of ascites of the patient.
Purified SAA has a molecular weight of 1200 by SDS-PAGE.
It showed a single band at 0 and did not react with other apolipoprotein antibodies. In addition, the amino acid sequence from the N terminus to Se
r Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala
Arg Asp Met Trp Arg Ala Tyr, which was reported from the database search by Dowlet et al. (Biochem.27: P1677,198).
It was found to be identical to form II and IV lacking N-terminal Arg according to 8).

1-2. Sensitization of anti-SAA antibody 1-1. Purified SAA obtained in 1-1
The rabbit was immunized after being mixed with an equal amount of an adjuvant and sufficiently emulsified. Immunization was performed every 2 weeks, and antiserum obtained by partially collecting blood after 4 months was subjected to 40% ammonium sulfate fractionation-dialysis-concentration to obtain anti-SAA antibody (0.01 mg / ml). This anti-S
Polystyrene latex (average particle size 0.11
(9 μm) at 37 ° C for 1 hour and then 0.1M
HEPES buffer was added and centrifuged (10,000 rp
After 20 minutes), the supernatant is discarded, and the precipitate is dispersed in a dispersion medium (0.1 M HEPES buffer solution containing 1% bovine serum albumin) to a final latex concentration of 0.4%.
It was suspended in 7.4) to obtain a reagent for latex aggregation reaction of SAA (hereinafter simply referred to as emulsion).

2. Measurement of Serum SAA 1 (Conventional Method) It was confirmed that a lot difference occurred in the conventional technique when 100-fold dilution was performed on 5 SAA high serum samples.
Three types of lots A, B, and C using antibodies having different lots and different immunization times were used as emulsions. The operation is as follows. Use 10 mM of each serum sample with 50 mM HEPES buffer (pH 7.4, hereinafter simply referred to as diluent).
It was diluted to 0 times and used as a measurement sample. Dispense 225 μl of the diluent and 20 μl of the measurement sample into the measurement cell, add 130 μl of the emulsion after 130 seconds, measure the first scattered light after 40 seconds (wavelength 660 nm), and then perform the second scattered light measurement after 130 seconds. The difference in scattered light intensity was determined. JP-A-4-25476
A calibration curve was set with a standard obtained by serially diluting human pool serum (SAA concentration 30 μg / ml) for which the SAA concentration was determined using HDL-SAA of JP-A-9 as a primary standard, and the SAA concentration was determined from the difference in scattered light intensity. The calibration curve was set for each reagent lot. Fully automatic immunochemical analyzer LX-3000 for measurement
(Eiken Chemical / Analytical Instruments, trade name) was used. The results are shown in Table 1. When the measured value of the reagent A is 100, the measured value of the reagent B always shows a low value of around 80, while the phenomenon that the measured value of the reagent C shows a high value of 120 to 150 is observed, and accurate measurement is possible. I confirmed that there is no.

[0022]

[Table 1]

3. Measurement of Serum SAA 2 (Method of the Present Invention) It was confirmed that, with respect to 5 serum samples having high SAA values, the present invention can suppress variations in the measured values between lots. The serum sample was diluted 10-fold with the same diluent as 1 and then further diluted 10-fold with 20-fold diluted human HDL to prepare a 100-fold diluted sample. By this operation, HDL was added to the final reaction solution so that HDL-C was 6.8 μg / ml. The HDL used was separated as the HDL by the following operation. The serum sample used in this example is different from 2. KBr was added to human pool serum at a specific gravity of 1.23 g / ml, dissolved, and then centrifuged (40000 rpm,
After 42 hours, the floating lipid fraction was collected. Purified water was added to the collected lipid fraction to adjust the specific gravity to 1.063 g / ml, and the mixture was centrifuged under the same conditions as the first time, and the lower layer fractions were collected to give purified HDL. Hitachi 85P-72 was used for centrifugation. The HDL used had an SAA content below the measurement range. The HDL amount was measured using HDL-C as an index. Serum H was used to measure the HDL-C content.
HDL-C555'Eiken 'for DL cholesterol measurement
(Manufactured by Eiken Chemical Co., Ltd.) was used. The results are shown in Table 2. HD is used for the immune reaction by using a diluent containing HDL.
By supplying L, it was possible to reduce the variation in the measured value between the reagent lots.

[0024]

[Table 2]

4. The following experiment was conducted in order to confirm in what concentration range the action of HDL confirmed by the amount of HDL used 3 was obtained. Serum samples were diluted 10-fold with diluents A) and B) with different HDL contents as shown in Table 3, and further diluted 10-fold with the diluent at the time of measurement to observe the effect of HDL concentration on the measured values. did. Human serum or human HDL used as a diluent
Used the SAA content below the measurement range.
Human serum, delipidated human serum, and purified HD used in the experiment
HDL-C contained in L is 30.4 mg / dl,
0.6 mg / dl, and 136.5 mg / dl. The compositions of A) and B) are summarized in Table 3. Serum sample is SA
One example of A high serum was used, and other operations were in accordance with 2. A) HDL-C content of human serum + delipidated human serum was 0-
30 mg / dl (0-as HDL-C in the final reaction solution)
1.8 μg / ml), or B) The HDL-C content was 5-50 with human HDL + buffer.
mg / dl (0.3-as HDL-C in the final reaction solution)
3.1 μg / ml)

[0026]

[Table 3] The results are shown in Tables 4 and 5. From these results, by adding human serum or human HDL to the place of the immune reaction, it is possible to suppress the variation in the measured value between the reagent lots. As for the effect of HDL, the variation of the measured value can be kept within 20% if the amount of HDL-C in the reaction solution is 0.3 μg / ml or more.

[0027]

[Table 4]

[0028]

[Table 5]

5. Measurement of Serum SAA 3 (Method of the Present Invention) It was confirmed that, with respect to 7 serum samples having high SAA values, the present invention can suppress variations in the measured values between lots. The serum sample was diluted 10-fold with the same diluent as 1 and further diluted 10-fold with 10-fold diluted human serum to prepare a 100-fold diluted sample. Reagents and measurement operations were in accordance with 2. Normal human serum was used as human serum, and HDL-C was 3 mg / dl. The serum sample used in this example is different from 2. By this operation, HDL- is contained in the final reaction solution.
This means that HDL was added so that C was 2 μg / ml. The results are shown in Table 6. It was confirmed that if HDL is supplied by adding human serum to the reaction system, fluctuations in measured values between reagent lots can be suppressed in the same manner as addition of purified HDL.

[0030]

[Table 6]

6. Serum SAA Measurement 4 (Method of the Present Invention) It was confirmed that, with respect to 4 serum samples having high SAA values, the present invention can suppress variations in the measured values between lots. A serum sample was diluted 10-fold with the same diluent as 1 and then further diluted 10-fold with 10-fold diluted animal serum to prepare a 100-fold diluted sample. Reagents and measurement operations were in accordance with 2. As animal sera, normal rabbit, goat, horse, bovine and porcine sera were used. The human serum or human HDL used in the diluent was S
Since it contains AA, it absorbs SAA from HDL and S
An AA content of less than the measurement range was used. Other animal sera and HDL were used as they are because they do not react with the SAA reagent used for the measurement. For comparison, a system containing 20-fold diluted human purified HDL was prepared. The serum sample used in this example is different from 2. The results are shown in Table 7.
The% in the table is a numerical value with 100 as the measured value when 1/20 human HDL is added. It was confirmed that if HDL is supplied by adding serum to the reaction system regardless of the type of animal, variation in measured values between reagent lots can be suppressed in the same manner as the addition of purified HDL. It was found that the same effect can be expected with animal serum, which can be easily procured, instead of human serum, which has a problem in supplying materials.

[0032]

[Table 7]

7. Serum SAA Measurement 5 (Method of the Present Invention) With respect to 5 serum samples having high SAA values, it was confirmed that the present invention can suppress variation in the measured values between lots. In all of the previous examples, HDL was added to the diluent side, but in this example, horse serum was added to the first reagent to confirm the effect.
The serum sample was diluted 100-fold with the same diluent as in 1 and horse serum was added to the diluent used as the first reagent, and the reagents and measurement operations were in accordance with 2. Horse serum was used to supply 0.7 μg / ml HDL as HDL-C to the immunoreaction field. The results are shown in Table 8. It was confirmed that the variation in the measured value between the reagent lots can also be suppressed by adding HDL to the first reagent. Ie SAA and HD
L It is not always necessary to incubate, and it can be said that it is sufficient to supply HDL immediately before the immune reaction and coexist in the place of the immune reaction.

[0034]

[Table 8]

8. Measurement of Serum SAA 6 (Method of the Present Invention) It was confirmed that the present invention improves the dilution linearity of a serum sample having a high SAA value. Serum samples are diluted 1-5 times with simple diluent or 10-fold diluted horse serum,
The AA concentration was measured. Other than the dilution operation, the reagents and operations were in accordance with 2. The results are shown in FIG. 1 (diluted solution) and FIG. 2 (horse serum). The dilution linearity is clearly improved when diluted with horse serum.

[Brief description of drawings]

FIG. 1 is a graph showing the dilution linearity of SAA obtained by a conventional method. Vertical axis shows SAA concentration (μg / m
l), the horizontal axis shows the dilution ratio (1 / n) of the serum sample.

FIG. 2 is a graph showing the dilution linearity of SAA obtained by the method of the present invention. Vertical axis shows SAA concentration (μg / m
l), the horizontal axis shows the dilution ratio (1 / n) of the serum sample.

Claims (11)

[Reference number] P-000303 [Claims] 1. A method for immunoassaying serum amyloid A, which comprises adding a high-density lipoprotein fraction to a reaction system in reacting serum amyloid A with an anti-serum amyloid A antibody. 2. The immunoassay method for serum amyloid A according to claim 1, wherein the serum amyloid A is contained in human blood. 3. The immunoassay method for serum amyloid A according to claim 1, wherein the high-density lipoprotein fraction is derived from an animal selected from the group consisting of human, horse, cow, and pig. 4. Immunization with serum amyloid A according to claim 1, wherein the measured value of the high-density lipoprotein fraction cholesterol of the high-density lipoprotein fraction to be added is 0.3 to 300 μg / ml or more in the reaction solution. Measurement method 5. The immunoassay method for serum amyloid A according to claim 4, wherein the measured value of the high density lipoprotein fraction cholesterol of the high density lipoprotein fraction to be added is 10 μg / ml or more in the reaction solution. 6. The immunoassay method for serum amyloid A according to claim 1, wherein a purified high-density lipoprotein fraction is added as the high-density lipoprotein fraction. 7. The immunoassay for serum amyloid A according to claim 1, wherein the antiserum amyloid A antibody is a polyclonal antibody or a monoclonal antibody. 8. The serum amyloid A according to claim 1, wherein the immunoassay is selected from the group consisting of radioimmunoassay, enzyme immunoassay, particle agglutination immunoassay, and immunoturbidimetric assay.
Immunoassay 9. An immunoassay in which serum amyloid A is reacted with an anti-serum amyloid A antibody, and serum amyloid A is measured based on the degree of binding between the two, and a high-density lipoprotein fraction is added to the reaction system. Method for suppressing fluctuation in measured value characterized by: 10. A reagent for immunological measurement of serum amyloid A, which comprises an antiserum amyloid A antibody and a high-density lipoprotein fraction. 11. An immunological assay reagent for serum amyloid A according to claim 10, wherein the antiserum amyloid A antibody is bound to an insoluble particle carrier.